Transcriptional activators regulate eukaryotic gene expression by stimulating the initiation of MRNA synthesis. A detailed knowledge of the initiation process is required for understanding the function of these factors. We propose to characterize the protein factors required, in addition to RNA polymerase II, for the accurate initiation of MRNA synthesis in yeast. Staring with a yeast whole cell extract, we will fractionate the transcription factors and study their interactions with the RNA polymerase, with each other, and with the DNA template. These factors, and their roles in transcriptional initiation, will be compared to those identified in mammalian and insect systems. Using the fractionated extract, we will study the process of transcriptional activation using several different yeast factors including GAL4, LEU3, HSF, ACE1, and STE12. Our goals will be to determine if different activators regulate different aspects of the transcriptional initiation process. The induction of transcription of the yeast galactose regulated gene is accompanied by phosphorylation of the GAL4 transcriptional activator. The phosphorylation appears to be required for activation based on both in vivo and in vitro experiments. We will study the amino acid sequence requirements for phosphorylation and the role of the modification in conversion of the GAL4/GAL80 complex to the transcriptionally active form. We will also use the non-phosphorylated form of the protein as a substrate to identify the kinase involved in induction. The phosphorylated and non-phosphorylated proteins will be studied using the fractionated transcription factors to determine how the modification affects initiation. The fractionated transcription factors will also be used to study a unique class of regulated nuclear genes. These genes share a DNA sequence element with the yeast mitochondrial transcription apparatus, and their expression is sensitive to the state of the mitochondrial genome. We will test the possibility that protein factors shared between the nucleus and the mitochondria are involved in co-ordinate gene regulation in the two genetic compartments.